Objective: To analyze the mechanism of the inhibition of IFNs induce SAMHD1 to hepatitis B virus(HBV)in liver cancer cells Huh7.0. Methods: (1) Recombiant expression plasmids of SAMHD1 (sterile alpha motif and histidine/ aspartic acid domain-containing protein 1) mutants that were defective in dNTPase (deoxynucleoside triphosphate triphosphohydrolase) activity and its nuclear location mutation were constructed. (2) The mRNA and protein levels of SAMHD1 in Huh7.0 cells by IFNs treatment were measured by real time and Western blot. (3) HBV core-associated DNA levels in transfected Huh7.0 cells were measured by Southern blot. (4) The location of SAMHD1 and its nuclear mutation in the Huh7.0 cells was measured by immunofluorescence. Results: (1) Both the RNA and protein levels of SAMHD1 were upregulated by interferons. (2) The antiviral function of SAMHD1 was lost when knocking down the expression of SAMHD1 by siRNA. (3) SAMHD1 lost its restriction towards HBV after mutating its NLS signal. Conclusion: IFNs can upregulate the expressing of SAMHD1 in Huh7 cells. SAMHD1 is mainly located in the nucleus and its restriction towards HBV dependent on its location.
T lymphocyte receptors (TCRs) play an important role in antigen recognition immune response, and their diversity is closely related to host immune response and tumor prognosis. Objective: To investigate the clonal diversity and cloning sequence of T cell receptor (TCR) in prostate cancer tissues and paracancerous tissues by high-throughput sequencing. Methods: The cancer tissues and paracancerous tissues of 5 patients with PC were collected.After DNA extraction, the CDR3 region of TCR β chain was amplified by multiplex PCR. Sequencing was performed with Illumina MiSeq, and the composition characteristics of the TCR CDR3 library of prostate cancer tissues were compared by data processing and comparison analysis. Results: Prostate cancer tissues had a higher degree of cloning and higher high-amplification clones than HECs (HEC: frequency>0.5% TCR clones of total sample reads) and obtained 24 pairs of differential expression in cancer tissue samples The VJ gene combination, 16 pairs of VJ gene combinations differentially expressed in paracancerous tissue samples. Conclusion: There are different V-J gene combinations and high cloned HEC in prostate cancer tissues and adjacent tissues, and cancer tissue samples have higher HEC. This study provides new data for immunological research on the development of PC, and provides reference for research on PC immune surveillance and T cell receptor mutation markers, laying a foundation for further research.
Objective: Effects of PKM2 knockdown on proliferation and apoptosis of human leukemia cells and its potential mechanism were investigated in our experiment. Methods: Lentivirus-based short hairpin RNA (shRNA) vector targeting PKM2 was transfected into K562 cells (shPKM2 group), and the vector-treated cells were named as the Vector group. mRNA and protein levels of PKM2 in K562 cells were determined by qRT-PCR and Western blot techniques, respectively. Cell proliferation activity was evaluated by CCK-8 assay in vitro. Cell cycle and apoptosis rate were analyzed by flow cytometry, the expression levels of apoptosis-related proteins Bax and Bcl-2 were measured by Western blot and the autophagy activity was measured by qRT-PCR and Western blot, respectively. Results: mRNA (t=11.58, P=0.000 3) and protein (t=11.88, P=0.000 3) levels of PKM2 were significantly decreased after PKM2 knockdown in K562 cells. In comparison to the Vector group, cell proliferation was inhibited by PKM2 deleption in shPKM2 group (F=118.87, P<0.000 1). Furthermore, G1-phase cell cycle was arrested, and the cell apoptosis rate was increased after PKM2 knockdown (t=37.23, P<0.000 1). Meanwhile, upregulated pro-apoptotic Bax protein levels (t=15.3, P=0.000 1) and downregulated anti-apoptotic Bcl-2 protein levels (t=9.965, P=0.000 6) were observed in shPKM2 group compared with the Vector group. In addition, decreased expression of PKM2 significantly downregulated LC3II levels (tLC3II=10.32, PLC3II=0.000 5) and elevated p62 levels (tp62=14.59, Pp62=0.000 1) in K562 cells. Finally, autophagy activator rapamycin rescued the inhibitory cell proliferation due to PKM2 knockdown (F=96.32, P<0.000 1). Conclusion: Above-mentioned results indicate that PKM2 knockdown can inhibit cell proliferation and promote cell apoptosis, at least partially through the cell autophagy, and PKM2 might be a potential target in the treatment of leukemia.
Human ribonuclease inhibitor (hRI) is an acidic pulping protein that regulates ribonuclease activity. By constructing a recombinant expression vector containing SUMO, IF2, GST, NusA, MsyB, Trx and MBP fusion tags, E.coli BL21 (DE3) was used as a host strain for auto-induction (AI) expression, thereby the expression level of hRI is improved. The expression of hRI was analyzed by MagNi magnetic bead purification and electrophoresis, and the protein with higher purity was obtained by RNase/Sepharose affinity chromatography.The concentration of the fusion protein obtained after purification was 2 960.513mg/L, which was compared with other companies, and its enzyme activity was about 50U/μl, which was successfully used for RNA protection. Purely provide a theoretic-al basis for the application of NusA-hRI.
Yarrowia lipolytica is a budding yeast and the budding position which determined the division plane is not selected randomly but determined by the distance of the last division site, that is, bud in the bipolar budding pattern. At present, the molecular regulation pathway of the bipolar budding is not well studied. In this report, the characterization of the vesicle protein YlSec15 was studied through the localization and overexpression. YlSec15 can locate inside the small buds and the neck of the middle or big buds, indicating the patterns of polar localization. Overexpression of YlSec15 in the yeast can inhibit the formation of hyphae and change budding pattern of cells, from bipolar to random. The causes of these changes may be due to the delocalization to the polar site of the excessive YlSec15. Besides, the normal localization of YlSec15 dependent on Ylras2. Based on this result, we speculate that YlSec15 may be involved in regulating the polar growth of cells through the signal pathway which is mediated by YlRas2. The discovery in this paper will enrich the molecular regulation mechanism of the bipolar budding and confirm the relationship between polar growth and vesicle transport in Yarrowia lipolytica.
Xylanase and mannanase are two important hemicellulases and feed enzyme preparations. Construction method of in vitro multimers in Pichia pastoris expression system has been used to construct recombinant plasmid pPICZαA/DSB-ManA which contained both DSB gene and ManA gene. Then, pPICZαA/DSB-ManA was transformed to host cells Pichia pastoris X33 by electroporation to obtain the co-expression recombinant strain X33/DSB-ManA. In shake-flask cultivation, xylanase activity and mannanase activity in the supernatant were 273.6 U/ml and 256.8 U/ml that reached 30.4% and 73.4% activity compared to X33/DSB and X33/ManA, respectively. Properties of the mixed enzyme suggested that the optimal reaction temperature for DSB and ManA is 75℃. Both xylanase and mannanase showed more than 60% activity across temperature ranges 45°C to 75°C. The optimal reaction pH value of DSB and ManA were 6.5 and 6.0, respectively. After incubation 1 hour under the pH3.0 and 40℃ treatment condition, mannanase activity of ManA displayed greater than 80% activity and xylanase activity of DSB revealed more than 50% activity. Meanwhile, DSB and ManA have shown good tolerance to a variety of metal ions and chemical reagents. Both enzyme activities could keep over 60% activity when the treatment concentration of different metal ions and chemical reagents was 1 millimole. Xylanase DSB and mannanase ManA have been co-expressed in Pichia pastoris successfully. The successful expression of the two enzymes of the recombinant strain could offer the theory basis for production and application of compound enzyme in the animal feed.
Objective: Interleukin (IL)-33 has important immunoregulatory effects and plays an important role in the disease. The aim of this paper is to achieve high-efficient expression of IL-33 in mammalian cells through gene optimization, and provide an important basis for disease mechanism research and vaccine immunoadjuvant application. Methods: The gene optimization for mammalian cell expression was carried out according to codon preference. The optimized and not optimized mIL-33 gene sequences were chemically synthesized. The human CD8αsignal peptide sequence was ligated to 5'end of the mIL-33 genes and led to fused CD8α+mIL-33 (Not optimized) and CD8α+mIL-33 (optimized) gene fragments by bridge PCR. The gene sequences of CD8α+ mIL-33 (Not optimized) or CD8α+mIL-33 (optimized) and EGFP expressing green fluorescent protein were respectively constructed into different expression units of plasmid pBudCE4.1 which has double expression units, and then 293FT cells were transfected with the recombinant plasmids using lipofectamine 3000 or PEI. The expression of recombinant proteins was detected by Western blot and ELISA. The expressed mIL-33s were used to stimulate macrophage Raw264.7, and the TNFα level of the culture supernatant was detected by ELISA to confirm the biological activity of expressed products. Results: The constructed recombinant plasmids were confirmed by restriction endonuclease digestion and sequencing analyses. The transfection efficiency of lipofectamine 3000 was higher than that of PEI. Western blot and ELISA showed higher levels of expression and secretion of IL-33 using optimized gene version. The expression level of mIL-33 in 293FT cells under the control of EF-1α promoter or CMV promoter was comparable. Expressed mIL-33 showed dose-dependent biological activity to stimulate TNF-α production in RAW264.7. Conclusion: The codon optimization significantly improved the secretory expression of mIL-33 with biological activity in mammalian cells, which laid a foundation for further research.
Alpha-ketoglutaric acid (α-KG), which is a keto acid product deaminated by glutamic acid, is widely used in food, medicine, fine chemicals and other fields as an important organic acid. To improve the yeild and the efficiency of biotransformation for the synthesis of α-ketoglutaric acid. First, by optimizing the conditions of whole-cell biocatalyst preparation and whole-cell biocatalysis conditions. Optimization conditions include the temperature, pH, inducer concentration, induction time in the whole-cell biocatalyst preparation process and the temperature, pH, biocatalyst concentration, biocatalytic time in the whole-cell biocatalysis process. Determine the optimal conditions of each item by detecting the amount of product α-KG. After the conditions were optimized, the maximum yield was increased by 54.9 % and the molar conversion was 39.6 %. Secondly, the directed evolution of L-amino acid deaminase by site-directed mutagenesis increased its catalytic ability. Through multiple mutations, screening, the yield of α-ketoglutaric acid biocatalytic synthesized by monosodium glutamate with optimal mutant E.coli BL21-pET-20b (+)-pm1152 was 100.9 g/L, and the molar conversion rate was 64.7 %, an increase of 66.3% compared to the control strain. The maximal yield and molar conversion of L-glutamic acid to α-KG was reached under the following optimal conditions: 20 g/L whole-cell biocatalyst, 30 ℃, pH 6.0, and 60-h biocatalysis, strain: E.coli BL21-pET-20b (+)-pm1152. The results showed that the conditional optimization and saturation mutation could effectively increase the whole-cell biocatalyst of recombinant E. coli to synthesize α-ketoglutaric acid.
A novel molecular imprinted electrochemical sensor was prepared by electro-polymerization on the surface of a glassy carbon electrode in NaAc-HAc buffer solution, and enrofloxacin was used as template molecular, o-phenylenediamine (OPD) and o-aminophenol (OAP) as composite functional monomer. The sensor could specifically recognize the template molecules and its analogues. In the experiment, 1 mol/L KCl and 5 mmol/L K3[Fe(CN)6] was used as characterization solution, the electrochemical sensor response characteristics were researched, and the preparation and detection conditions of the sensor were optimized by square wave voltammetry (SWV) and cyclic voltammetry (CV). The results showed that under the optimal conditions, the linear relationship between SWV peak current difference and the concentrations of enrofloxacin was found in the range of 2×10 -6 mol/L4×10 -5 mol/L with the linear correlation coefficient ( R 2 ) of 0.9990, the detection limit was 7.0×10 -7mol/L, and the sensor had good stability, repeatability and good selectivity to enrofloxacin and its analogues. Enrofloxacin in real samples, such as milk, egg, chicken and pork, was detected by the developed sensor, the average recoveries were between 83.2% and 92.7%, and the relative standard deviations ( RSDs ) were 1.0%~4.8% ( n=5 ). This sensor is simple, sensitive, low cost, and has good selectivity and stability, which provide it a good application prospect.
Objective: To construct the recombinant Bacillus subtilis with D-hydantoinase(DHase, hyd ene) and D-carbamoylase(DCase, adc gene) activity as catalyst to produce D-p-hydroxyphenylglycine(D-HPG) by hydantoinase process. Methods: The hyd gene expression plasmids were constructed. The effects of divalent metal ions in medium on the DHase activity was investigated. The acoR gene was over-expressed to investigate the correlation between the activator protein AcoR and PacoA-hyd gene expression. The optimal promoter used to express adc gene was screened from the PAE, PspoVG, Pcdd and PlytR. The hyd and adc gene co-expression plasmid was constructed and its catalytic properties was characterized. Results: The hyd gene expression plasmid pHPS and pUBS were successfully constructed. Mn 2+ has a strong activation effect on DHase and the activity of the 168N/pUBS reached 956U/gDCW when 0.8mmol/L MnCl2·4H2O was added to the medium. Adding a copy of the Pcdd-acoR gene, DHase activity of the LSL02/pUBS reached 1 470 U/gDCW. The LN04 strain integrated with the PAE-adc had the highest DCase activity. The co-expression plasmid pUBSC was constructed, and under the optimum conditions of pH 8.0 and 40℃, with initial substrate concentration of 20g/L, the catalytic activity of LSL02/pUBSC could last for 12h to generate 14.32g/L of D-HPG with the conversion rate of 95%, yield of 82.4%. Conclusion: The recombinant strain with higher dual enzyme activity can be obtained when heterologous hyd and adc were expressed in Bacillus subtilis and it is technically feasible and has the application prospect for preparing D-HPG by hydantoinase process.
Optogenetics is an emerging biological technique that combines optics and genetics to precisely control specific cells within organisms. Since the application of microbial opsins, optogenetics has gained substantial progresses in discovering opsins, optic-control methods and genetic strategies that are based on viruses and recombinases. Optogenetics apply widely in modern neuroscience, playing important roles in the study of neural circuits and behavior, the pathological mechanism of various central nervous system diseases and psychiatric disorders. This review summarizes the development of optogenetic techniques, meanwhile emphasizes the latest advances in the opsin exploration and localized expression, aiming to provide references for research in optogenetics and related fields.
Diseases such as dengue fever caused by denugue virus lead to considerable economic, medical and social burdens every year, which seriously threaten the health of human life. Among the various dengue virus vaccines currently under study, the 3'UTRΔ30 series of live attenuated vaccines prepared by reverse genetics have shown good protection in clinical trials because of their good immunogenicity, high titer and low cost. As a consequence, research on this kind of vaccine advances fast. The 3'UTRΔ30 quadruple vaccine, one of the most promising dengue attenuated inactivated vaccines today, which can produce balanced immune protection against the four serotypes of dengue virus, is in phase III clinical trials, showing strong efficacy and few adverse reactions. It is expected to be on the market after the end of the follow-up period. In order to gain a deeper understanding of the 3'UTRΔ30 series vaccines, the origin, preparation methods and clinical research are mainly focused on.
In the current environment of biopharmaceuticals, cost pressures, rapidly fluctuating market demands and growing competition among biosimilars, existing bio-manufacturing technologies are challenged, and so that biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. As one of the important processes in mammalian cell culture, perfusion culture has two advantages. Firstly, it can provide a stable environment favorable to cells by continuously removing by-products and adding nutrients, so that it can solve the problems of unstable protein amount or low expression level. Also, it can optimize capacity utilization and increase production efficiency by increasing volumetric productivity. This paper systematically reviewed the progress of perfusion culture for mammalian cell culture,and it provides reference for further development and application.
Monoclonal antibodies have played an inestimable role in the treatment of cancer and autoimmune diseases with their unique mechanism and high efficiency, which have become the focus of research in biopharmaceutics. In this paper, all the data was extracted from Clarivate via Cortellis. Through the combination of quantitative data analysis and expert wisdom, the competitive landscape of monoclonal antibodies was presented from various dimensions, including the overall trend of R&D and industrialization, focus countries, technology and drug categories, targets, market share and commercial deal. The results showed that among the 133 kinds of launched monoclonal antibodies recently, 37.6% of them belonged to humanized monoclonal antibodies. HER, TNF, CD20, PD-1/L1, VEGF and CD3 were the “hot” antibody target molecule, of the total number of target-based actions which was the most numerous in HER2 tyrosine kinase receptor inhibitor. The total volume of monoclonal antibodies in China ranked second to the United States. But there were only 8 monoclonal antibodies approved in China, which was far below the level of United States. There were 22 launched monoclonal antibodies achieved annual sales over $1 biliion in 2017. Access to 1408 monoclonal antibodies deal records, drug development/commercial license was the primary agreement type. In the future, the development trend of monoclonal antibodies will be focused on new targets, new indications and new therapeutic schemes, and then more “blockbuster drugs” will be produced.
